PROJECT SUMMARY ABSTRACT Clostridioides (Clostridium) difficile infection (CDI) is a common healthcare-associated infection in US children and adults. CDI, which ranges from mild diarrhea to life-threatening colitis, is associated with substantial morbidity, mortality, and excess healthcare expenditures. The CDC has classified C. difficile as an ?immediate public health threat that requires urgent and aggressive action.? This call to action has expanded CDI investigation and prompted efforts to develop novel and more effective prevention strategies. Immunological agents are a promising strategy for CDI prevention; a monoclonal antibody against C. difficile is recently FDA approved for CDI prevention, and several toxin-based vaccines are in clinical development. Despite these products showing promise for CDI prevention, many patients have failed these therapies. The immunizations currently under clinical investigation were developed based on knowledge of C. difficile immunogenicity in animals, which may not adequately represent C. difficile immunogenicity in humans. To better understand the relative immunogenicity of C. difficile antigens in humans, we propose characterization of the plasmablast response following natural CDI. Plasmablasts are plasma cell precursors that produce antibodies directed against a pathogen; antigen-specific plasmablasts can be isolated from peripheral blood 1-3 weeks after infection. Antibodies encoded by these cells can be produced in vitro and the target antigens identified. This innovative method has been recently successfully applied to other infectious diseases, including to Kawasaki Disease by our research team. By analyzing the plasmablast response following CDI, we will identify the breadth of the human antibody response to C. difficile infection. We hypothesize that the antibody response to C. difficile is not limited to toxin antigens, and that antibodies against both toxin and non-toxin antigens provide protection against CDI. Specifically, in this feasibility study, we aim to identify and clone the peripheral blood plasmablast response following CDI in 3 children and 3 adults, and identify C. difficile antigenic targets of oligoclonal post-CDI plasmablasts using various proteomics analyses. Knowledge gained in this feasibility study will inform design of larger studies to validate immunogenicity of C. difficile antigens in a more diverse patient cohort and the neutralizing ability of these antibodies across a diverse set of C. difficile strain types. In future studies, we will determine the specific toxin and non-toxin epitopes recognized by antibodies following natural CDI in humans, and we will study how antibody responses to C. difficile toxin and non-toxin antigens impact C. difficile colonization and infection. Identification of broadly immunogenic antigens will guide future work to investigate potential immunization candidates to prevent CDI and colonization. Our research team and the Northwestern University academic environment possess the skills and resources, respectively, that are necessary for successful completion of the proposed studies, including assembly of patient cohort, bacterial genomics, plasmablast isolation, plasmablast antibody synthesis, and proteomics. !